Nitrogen Redox Metabolism of a Heterotrophic, Nitrifying-Denitrifying Alcaligenes sp. from Soil

Metabolic characteristics of a heterotrophic, nitrifier-denitrifier Alcaligenes sp. isolated from soil were further characterized. Pyruvic oxime and hydroxylamine were oxidized to nitrite aerobically by nitrification-adapted cells with specific activities (V_max) of 0.066 and 0.003 μmol of N × min⁻¹ × mg of protein⁻¹, respectively, at 22°C. K_m values were 15 and 42 μM for pyruvic oxime and hydroxylamine, respectively. The greater pyruvic oxime oxidation activity relative to hydroxylamine oxidation activity indicates that pyruvic oxime was a specific substrate and was not oxidized appreciably via its hydrolysis product, hydroxylamine. When grown as a denitrifier on nitrate, the bacterium could not aerobically oxidize pyruvic oxime or hydroxylamine to nitrite. However, hydroxylamine was converted to nearly equimolar amounts of ammonium ion and nitrous oxide, and the nature of this reaction is discussed. Cells grown as heterotrophic nitrifiers on pyruvic oxime contained two enzymes of denitrification, nitrate reductase and nitric oxide reductase. The nitrate reductase was the dissimilatory type, as evidenced by its extreme sensitivity to inhibition by azide and by its ability to be reversibly inhibited by oxygen. Cells grown aerobically on organic carbon sources other than pyruvic oxime contained none of the denitrifying enzymes surveyed but were able to oxidize pyruvic oxime to nitrite and reduce hydroxylamine to ammonium ion.     

Methane Oxidation by Nitrosococcus oceanus and Nitrosomonas europaea

Chemolithotrophic ammonium-oxidizing and nitrite-oxidizing bacteria including Nitrosomonas europaea, Nitrosococcus oceanus, Nitrobacter sp., Nitiospina gracilis, and Nitrococcus mobilis were examined as to their ability to oxidize methane in the absence of ammonium or nitrite. All ammonium oxidizers tested had the ability to oxidize significant amounts of methane to CO₂ and incorporate various amounts into cellular components. None of the nitrite-oxidizing bacteria were capable of methane oxidation. The methane-oxidizing capabilities of Nitrosococcus oceanus and Nitrosomonas europaea were examined with respect to ammonium and methane concentrations, nitrogen source, and pH. The addition of ammonium stimulated both CO₂ production and cellular incorporation of methane-carbon by both organisms. Less than 0.1 mM CH₄ in solution inhibited the oxidation of ammonium by Nitrosococcus oceanus by 87%. Methane concentrations up to 1.0 mM had no inhibitory effects on ammonium oxidation by Nitrosomonas europaea. In the absence of NH₄-N, Nitrosococcus oceanus achieved a maximum methane oxidation rate of 2.20 × 10⁻² μmol of CH₄ h⁻¹ mg (dry weight) of cells⁻¹, which remained constant as the methane concentration was increased. In the presence of NH₄-N (10 ppm [10 μg/ml]), its maximum rate was 26.4 × 10⁻² μmol of CH₄ h⁻¹ mg (dry weight) of cells⁻¹ at a methane concentration of 1.19 × 10⁻² mM. Increasing the methane concentration above this level decreased CO₂ production, whereas cellular incorporation of methane-carbon continued to increase. Nitrosomonas europaea showed a linear response throughout the test range, with an activity of 196.0 × 10⁻² μmol of CH₄ h⁻¹ mg (dry weight) of cells ⁻¹ at a methane concentration of 1.38 × 10⁻¹ mM. Both nitrite and nitrate stimulated the oxidation of methane. The pH range was similar to that for ammonium oxidation, but the points of maximum activity were at lower values for the oxidation of methane.     

Effect of Low Sulfate Concentrations on Lactate Oxidation and Isotope Fractionation during Sulfate Reduction by Archaeoglobus fulgidus Strain Z

The effect of low substrate concentrations on the metabolic pathway and sulfur isotope fractionation during sulfate reduction was investigated for Archaeoglobus fulgidus strain Z. This archaeon was grown in a chemostat with sulfate concentrations between 0.3 mM and 14 mM at 80°C and with lactate as the limiting substrate. During sulfate reduction, lactate was oxidized to acetate, formate, and CO₂. This is the first time that the production of formate has been reported for A. fulgidus. The stoichiometry of the catabolic reaction was strongly dependent on the sulfate concentration. At concentrations of more than 300 μM, 1 mol of sulfate was reduced during the consumption of 1 mol of lactate, whereas only 0.6 mol of sulfate was consumed per mol of lactate oxidized at a sulfate concentration of 300 μM. Furthermore, at low sulfate concentrations acetate was the main carbon product, in contrast to the CO₂ produced at high concentrations. We suggest different pathways for lactate oxidation by A. fulgidus at high and low sulfate concentrations. At about 300 μM sulfate both the growth yield and the isotope fractionation were limited by sulfate, whereas the sulfate reduction rate was not limited by sulfate. We suggest that the cell channels more energy for sulfate uptake at sulfate concentrations below 300 to 400 μM than it does at higher concentrations. This could explain the shift in the metabolic pathway and the reduced growth yield and isotope fractionation at low sulfate levels.     

Estimation of the fructose diphosphatase-phosphofructokinase substrate cycle in the flight muscle of Bombus affinis

1. Substrate cycling of fructose 6-phosphate through reactions catalysed by phosphofructokinase and fructose diphosphatase was estimated in bumble-bee (Bombus affinis) flight muscle in vivo. 2. Estimations of substrate cycling of fructose 6-phosphate and of glycolysis were made from the equilibrium value of the ³H/¹⁴C ratio in glucose 6-phosphate as well as the rate of ³H release to water after the metabolism of [5-³H,U-¹⁴C]glucose. 3. In flight, the metabolism of glucose proceeded exclusively through glycolysis (20.4μmol/min per g fresh wt.) and there was no evidence for substrate cycling. 4. In the resting bumble-bee exposed to low temperatures (5°C), the pattern of glucose metabolism in the flight muscle was altered so that substrate cycling was high (10.4μmol/min per g fresh wt.) and glycolysis was decreased (5.8μmol/min per g fresh wt.). 5. The rate of substrate cycling in the resting bumble-bee flight muscle was inversely related to the ambient temperature, since at 27°, 21° and 5°C the rates of substrate cycling were 0, 0.48 and 10.4μmol/min per g fresh wt. respectively. 6. Calcium ions inhibited fructose diphosphatase of the bumble-bee flight muscle at concentrations that were without effect on phosphofructokinase. The inhibition was reversed by the presence of a Ca²⁺-chelating compound. It is proposed that the rate of fructose 6-phosphate substrate cycling could be regulated by changes in the sarcoplasmic Ca²⁺ concentration associated with the contractile process.     

Aerosols of Mycoplasmas, L Forms, and Bacteria: Comparison of Particle Size, Viability, and Lethality of Ultraviolet Radiation

Aerosols of microorganisms were tested for particle size by use of an Andersen sampler. Mycoplasma aerosols had an average count median diameter (CMD) of 2.1 ± 0.5 μ. Staphylococcus aureus L forms gave an average CMD of 4.6 ± 1.7 μ; the diphtheroid L form, a CMD of 3.4 ± 0.3 μ. Escherichia coli had a CMD of 5.4 ± 2.5 μ; Neisseria sicca, 3.3 ± 0.5 μ; N. meningitidis, 3.4 ± 0.2 μ. S. aureus ATCC 6538, the parent strain of the L form, yielded a CMD of 3.9 ± 1.2 μ. Candida albicans gave an average CMD of 5.9 ± 1.4 μ. All organisms tested survived aerosolizing and could be recovered in viable form for at least 1 hr. Ultraviolet radiation at 2,537 A destroyed the bacteria and mycoplasmas instantaneously, and destroyed 87% of the L forms of S. aureus, 69% of the diphtheroid L form, and 98% of the C. albicans cells. After irradiation, viable particles of the L form and C. albicans aerosols were consistently larger, indicating that clumping led to survival. Submicron size particles were found in aerosols of all species tested except C. albicans.     

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L⁻¹ for NO₃ ⁻ and NH₄ ⁺, 0.5–50 μmol N L⁻¹ for urea, 0.5–100 μmol N L⁻¹ for glu and cyanate, and 0.5–200 μmol N L⁻¹ for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μ_m, 1.51±0.06 d⁻¹ and 1.50±0.05 d⁻¹, respectively). However, cultures grown on cyanate, NO₃ ⁻, and NH₄ ⁺ had lower half saturation constants (K_μ, 0.28–0.51 μmol N L⁻¹). N uptake kinetics were measured in NO₃ ⁻-deplete and -replete batch cultures of P. donghaiense. In NO₃ ⁻-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO₃ ⁻, NH₄ ⁺, urea and algal amino acids; uptake was saturated at or below 50 μmol N L⁻¹. In NO₃ ⁻-replete batch cultures, NH₄ ⁺, urea, and algal amino acid uptake kinetics were similar to those measured in NO₃ ⁻-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.     

Detection of Phosphatidylcholine-Coated Gold Nanoparticles in Orthotopic Pancreatic Adenocarcinoma using Hyperspectral Imaging

Nanoparticle uptake and distribution to solid tumors are limited by reticuloendothelial system systemic filtering and transport limitations induced by irregular intra-tumoral vascularization. Although vascular enhanced permeability and retention can aid targeting, high interstitial fluid pressure and dense extracellular matrix may hinder local penetration. Extravascular diffusivity depends upon nanoparticle size, surface modifications, and tissue vascularization. Gold nanoparticles functionalized with biologically-compatible layers may achieve improved uptake and distribution while enabling cytotoxicity through synergistic combination of chemotherapy and thermal ablation. Evaluation of nanoparticle uptake in vivo remains difficult, as detection methods are limited. We employ hyperspectral imaging of histology sections to analyze uptake and distribution of phosphatidylcholine-coated citrate gold nanoparticles (CGN) and silica-gold nanoshells (SGN) after tail-vein injection in mice bearing orthotopic pancreatic adenocarcinoma. For CGN, the liver and tumor showed 26.5±8.2 and 23.3±4.1 particles/100μm² within 10μm from the nearest source and few nanoparticles beyond 50μm, respectively. The spleen had 35.5±9.3 particles/100μm² within 10μm with penetration also limited to 50μm. For SGN, the liver showed 31.1±4.1 particles/100μm² within 10μm of the nearest source with penetration hindered beyond 30μm. The spleen and tumor showed uptake of 22.1±6.2 and 15.8±6.1 particles/100μm² within 10μm, respectively, with penetration similarly hindered. CGH average concentration (nanoparticles/μm²) was 1.09±0.14 in the liver, 0.74±0.12 in the spleen, and 0.43±0.07 in the tumor. SGN average concentration (nanoparticles/μm²) was 0.43±0.07 in the liver, 0.30±0.06 in the spleen, and 0.20±0.04 in the tumor. Hyperspectral imaging of histology sections enables analysis of phosphatidylcholine-coated gold-based nanoparticles in pancreatic tumors with the goal to improve nanotherapeutic efficacy.     

4-Methylumbelliferyl-β-N-Acetylglucosaminide Hydrolysis by a High-Affinity Enzyme, a Putative Marker of Protozoan Bacterivory

Hydrolysis of an artificial fluorogenic substrate, 4-methylumbelliferyl-β-N-acetylglucosaminide, has been studied in a monoculture predator-prey system with either a flagellate (Bodo saltans) or a ciliate (Cyclidium sp.) fed upon pure bacterial culture (Aeromonas hydrophila or Alcaligenes xylosoxidans). Aeromonas hydrophila produced a low-affinity β-N-acetylglucosaminidase-like enzyme (K_m, 100 μmol liter^-1) but Alcaligenes xylosoxidans did not. Inoculation of both bacterial strains with bacterivorous protozoa induced the occurrence of another, high-affinity, β-N-acetylglucosaminidase-like enzyme (K_m, <0.5 μmol liter^-1). The latter enzyme showed significant, close correlations with total grazing rates of both B. saltans (r² = 0.96) and Cyclidium sp. (r² = 0.89) estimated by using uptake of fluorescently labelled bacteria. Further significant correlations between several protozoan parameters and kinetic parameters of this enzyme suggest its likely protozoan origin. If both types of enzyme occurred together, they could be satisfactorily distinguished by using kinetic data analysis. Hence, measurements of β-N-acetylglucosaminidase-like activities might be promising to use to improve estimations of protozoan bacterivory.     

Effect of Soil Ammonium Concentration on N2O Release and on the Community Structure of Ammonia Oxidizers and Denitrifiers

The effect of ammonium addition (6.5, 58, and 395 μg of NH₄⁺-N g [dry weight] of soil⁻¹) on soil microbial communities was explored. For medium and high ammonium concentrations, increased N₂O release rates and a shift toward a higher contribution of nitrification to N₂O release occurred after incubation for 5 days at 4°C. Communities of ammonia oxidizers were assayed after 4 weeks of incubation by denaturant gradient gel electrophoresis (DGGE) of the amoA gene coding for the small subunit of ammonia monooxygenase. The DGGE fingerprints were invariably the same whether the soil was untreated or incubated with low, medium, or high ammonium concentrations. Phylogenetic analysis of cloned PCR products from excised DGGE bands detected amoA sequences which probably belonged to Nitrosospira 16S rRNA clusters 3 and 4. Additional clones clustered with Nitrosospira sp. strains Ka3 and Ka4 and within an amoA cluster from unknown species. A Nitrosomonas-like amoA gene was detected in only one clone. In agreement with the amoA results, community profiles of total bacteria analyzed by terminal restriction fragment length polymorphism (T-RFLP) showed only minor differences. However, a community shift occurred for denitrifier populations based on T-RFLP analysis of nirK genes encoding copper-containing nitrite reductase with incubation at medium and high ammonia concentrations. Major terminal restriction fragments observed in environmental samples were further described by correspondence to cloned nirK genes from the same soil. Phylogenetic analysis grouped these clones into clusters of soil nirK genes. However, some clones were also closely related to genes from known denitrifiers. The shift in the denitrifier community was probably the consequence of the increased supply of oxidized nitrogen through nitrification. Nitrification activity increased upon addition of ammonium, but the community structure of ammonium oxidizers did not change.     

Isolation of a Multiheme Protein with Features of a Hydrazine-Oxidizing Enzyme from an Anaerobic Ammonium-Oxidizing Enrichment Culture

A multiheme protein having hydrazine-oxidizing activity was purified from enriched culture from a reactor in which an anammox bacterium, strain KSU-1, was dominant. The enzyme has oxidizing activity toward hydrazine but not hydroxylamine and is a 130-kDa homodimer composed of a 62-kDa polypeptide containing eight hemes. It was therefore named hydrazine-oxidizing enzyme (HZO). With cytochrome c as an electron acceptor, the V_max and K_m for hydrazine are 6.2 ± 0.3 μmol/min · mg and 5.5 ± 0.6 μM, respectively. Hydrazine (25 μM) induced an increase in the proportion of reduced form in the spectrum, whereas hydroxylamine (500 μM) did not. Two genes coding for HZO, hzoA and hzoB, were identified within the metagenomic DNA from the culture. The genes encode the same amino acid sequence except for two residues. The sequences deduced from these genes showed low-level identities (<30%) to those of all of the hydroxylamine oxidoreductases reported but are highly homologous to two hao genes found by sequencing the genome of “Candidatus Kuenenia stuttgartiensis” (88% and 89% identities). The purified enzyme might therefore be a novel hydrazine-oxidizing enzyme having a critical role in anaerobic ammonium oxidation.     

Modeling Reduction of Uranium U(VI) under Variable Sulfate Concentrations by Sulfate-Reducing Bacteria

The kinetics for the reduction of sulfate alone and for concurrent uranium [U(VI)] and sulfate reduction, by mixed and pure cultures of sulfate-reducing bacteria (SRB) at 21 ± 3°C were studied. The mixed culture contained the SRB Desulfovibrio vulgaris along with a Clostridium sp. determined via 16S ribosomal DNA analysis. The pure culture was Desulfovibrio desulfuricans (ATCC 7757). A zero-order model best fit the data for the reduction of sulfate from 0.1 to 10 mM. A lag time occurred below cell concentrations of 0.1 mg (dry weight) of cells/ml. For the mixed culture, average values for the maximum specific reaction rate, V_max, ranged from 2.4 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h⁻¹) at 0.25 mM sulfate to 5.0 ± 1.1 μmol of sulfate/mg (dry weight) of SRB · h⁻¹ at 10 mM sulfate (average cell concentration, 0.52 mg [dry weight]/ml). For the pure culture, V_max was 1.6 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h⁻¹ at 1 mM sulfate (0.29 mg [dry weight] of cells/ml). When both electron acceptors were present, sulfate reduction remained zero order for both cultures, while uranium reduction was first order, with rate constants of 0.071 ± 0.003 mg (dry weight) of cells/ml · min⁻¹ for the mixed culture and 0.137 ± 0.016 mg (dry weight) of cells/ml · min⁻¹ (U₀ = 1 mM) for the D. desulfuricans culture. Both cultures exhibited a faster rate of uranium reduction in the presence of sulfate and no lag time until the onset of U reduction in contrast to U alone. This kinetics information can be used to design an SRB-dominated biotreatment scheme for the removal of U(VI) from an aqueous source.     

Metabolism of propionate by sheep liver. Oxidation of propionate by homogenates

1. The rate and stability to aging of the metabolism of propionate by sheep-liver slices and sucrose homogenates were examined. Aging for up to 20min. at 37° in the absence of added substrate had little effect with slices, whole homogenates or homogenates without the nuclear fraction. 2. Metabolism of propionate by sucrose homogenates was confined to the mitochondrial fraction, but the mitochondrial supernatant (microsomes plus cell sap) stimulated propionate removal. 3. The rate of propionate metabolism by liver slices was higher in a high potassium phosphate–bicarbonate medium [0·88(±s.e.m. 0·16)μmole/mg. of N/hr.] than in Krebs–Ringer bicarbonate medium [0·44(±s.e.m. 0·13)μmole/mg. of N/hr.]. 4. Metabolism of propionate by sucrose homogenates freed from nuclei was dependent on the presence of oxygen, carbon dioxide and ATP. Propionate removal was stimulated 250% by Mg²⁺ ions and 670% by cytochrome c. 5. In the complete medium 2·39(±s.e.m. 0·15)μmoles of propionate were consumed/mg. of N/hr. 6. The ratio of oxygen consumption to propionate utilization was sufficient to account for the complete oxidation of half the propionate consumed. 7. The only products detected under these conditions were succinate, fumarate and malate. Propionate had no effect on the production of lactate from endogenous sources and did not itself give rise to lactate. 8. Methylmalonate did not accumulate when propionate was metabolized and was not oxidized. It was detected as an intermediate in the conversion of propionyl-CoA into succinate. The rate of this reaction sequence was adequate to account for the rate of propionate metabolism by sucrose homogenates or slices, provided that the rate of formation of propionyl-CoA was not limiting. 9. The methylmalonate pathway was predominantly a mitochondrial function. 10. The metabolism of propionate appeared to be dependent on active oxidative phosphorylation.     

Biodegradation of Chloromethane by Pseudomonas aeruginosa Strain NB1 under Nitrate-Reducing and Aerobic Conditions

Pseudomonas aeruginosa strain NB1 uses chloromethane (CM) as its sole source of carbon and energy under nitrate-reducing and aerobic conditions. The observed yield of NB1 was 0.20 (±0.06) (mean ± standard deviation) and 0.28 (±0.01) mg of total suspended solids (TSS) mg of CM⁻¹ under anoxic and aerobic conditions, respectively. The stoichiometry of nitrate consumption was 0.75 (±0.10) electron equivalents (eeq) of NO₃⁻ per eeq of CM, which is consistent with the yield when it is expressed on an eeq basis. Nitrate was stoichiometrically converted to dinitrogen (0.51 ± 0.05 mol of N₂ per mol of NO₃⁻). The stoichiometry of oxygen use with CM (0.85 ± 0.21 eeq of O₂ per eeq of CM) was also consistent with the aerobic yield. Stoichiometric release of chloride and minimal accumulation of soluble metabolic products (measured as chemical oxygen demand) following CM consumption, under anoxic and aerobic conditions, indicated complete biodegradation of CM. Acetylene did not inhibit CM use under aerobic conditions, implying that a monooxygenase was not involved in initiating aerobic CM metabolism. Under anoxic conditions, the maximum specific CM utilization rate (k) for NB1 was 5.01 (±0.06) μmol of CM mg of TSS⁻¹ day⁻¹, the maximum specific growth rate (μ_max) was 0.0506 day⁻¹, and the Monod half-saturation coefficient (K_s) was 0.067 (±0.004) μM. Under aerobic conditions, the values for k, μ_max, and K_s were 10.7 (±0.11) μmol of CM mg of TSS⁻¹ day⁻¹, 0.145 day⁻¹, and 0.93 (±0.042) μM, respectively, indicating that NB1 used CM faster under aerobic conditions. Strain NB1 also grew on methanol, ethanol, and acetate under denitrifying and aerobic conditions, but not on methane, formate, or dichloromethane.     

Steady-State Effects of 2,5,2′,5′-Tetrachlorobiphenyl on Growth, Photosynthesis, and P Uptake in Selenastrum capricornutum

The steady-state effect of 2,5,2′,5′-tetrachlorobiphenyl (TCBP) on the green alga Selenastrum capricornutum was investigated in a P-limited two-stage chemostat system. The partition coefficient of this polychlorinated biphenyl congener was 5.9 × 10⁴ in steady-state cultures. At a cellular TCBP concentration of 12.2 × 10⁻⁸ ng · cell⁻¹, growth rate was not affected. However, photosynthetic capacity (P_max) was significantly enhanced by TCBP (56 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ versus 34 × 10⁻⁹ μmol of C · cell⁻¹ · h⁻¹ in the control). Photosynthetic efficiency, or the slope of the photosynthesis-irradiance curve, was also significantly higher. There was little difference in the cell chlorophyll a content, and therefore the difference in these photosynthetic characteristics was the same even when they were expressed on a per-chlorophyll a basis. Cell C content was higher in TCBP-containing cells than in TCBP-free cells, but approximately 36% of the C fixed by cells with TCBP was not incorporated as cell C. The maximum P uptake rate was also enhanced by TCBP, but the half-saturation concentration appeared to be unaffected.     

Paurodontella parapitica n. sp. (Nematoda: Hexatylina,Sphaerularioidea) from Kermanshah Province,Western Iran

Paurodontella parapitica n. sp., collected from the rhizosphere of an apple tree in Kermanshah province, western Iran, is described. The new species is characterized by a body length of 505 to 723 µm (females) and 480 to 600 µm (males), lip region continuous by depression; 7 to 8 μm broad, 3 to 4 µm high, stylet length 7 to 9 µm or 1 to 1.3 times the lip region diameter, short postuterine sac of 4 to 6 μm long, lateral fields with five to six incisures; outer incisures crenated and inner incisures weakly crenated, excretory pore situated 90 to 100 µm from anterior end; functional males common in the population, with spicules 24 to 26 μm long. Tail of both sexes similar, almost straight and elongate-conoid. The new species resembles in morphology and morphometrics to four known species of the genus, namely P. apitica, P. minuta, P. myceliophaga, and P. sohailai. The results of phylogenetic analyses based on sequences of D2/D3 expansion region of 28S rRNA gene revealed this genus is polyphyletic in four different clades in Tylenchid.     

Alpha-tocopherol is essential for acquired chill-light tolerance in the cyanobacterium Synechocystis sp. strain PCC 6803

Unlike Escherichia coli, the cyanobacterium Synechocystis sp. strain PCC 6803 is insensitive to chill (5°C) in the dark but rapidly losses viability when exposed to chill in the light (100 μmol photons m⁻² s⁻¹). Preconditioning at a low temperature (15°C) greatly enhances the chill-light tolerance of Synechocystis sp. strain PCC 6803. This phenomenon is called acquired chill-light tolerance (ACLT). Preconditioned wild-type cells maintained a substantially higher level of α-tocopherol after exposure to chill-light stress. Mutants unable to synthesize α-tocopherol, such as slr1736, slr1737, slr0089, and slr0090 mutants, almost completely lost ACLT. When exposed to chill without light, these mutants showed no or a slight difference from the wild type. When complemented, the slr0089 mutant regained its ACLT. Copper-regulated expression of slr0090 from P_petE controlled the level of α-tocopherol and ACLT. We conclude that α-tocopherol is essential for ACLT of Synechocystis sp. strain PCC 6803. The role of α-tocopherol in ACLT may be based largely on a nonantioxidant activity that is not possessed by other tocopherols or pathway intermediates.     

Microscale Diversity of the Genus Nitrobacter in Soil on the Basis of Analysis of Genes Encoding rRNA

We looked at the diversity of NO₂⁻ oxidizers at field scale by examining isolates at clump scale and in microsamples of soil (diameter, 50 μm). The genetic distances (as determined by amplified ribosomal DNA restriction analysis performed with Nitrobacter-specific primers) in a small clump of soil were as large as those between reference strains from large geographical areas. Diversity in individual microsamples was shown by serotyping.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.     

Protein Engineering of Epoxide Hydrolase from Agrobacterium radiobacter AD1 for Enhanced Activity and Enantioselective Production of (R)-1-Phenylethane-1,2-Diol

DNA shuffling and saturation mutagenesis of positions F108, L190, I219, D235, and C248 were used to generate variants of the epoxide hydrolase of Agrobacterium radiobacter AD1 (EchA) with enhanced enantioselectivity and activity for styrene oxide and enhanced activity for 1,2-epoxyhexane and epoxypropane. EchA variant I219F has more than fivefold-enhanced enantioselectivity toward racemic styrene oxide, with the enantiomeric ratio value (E value) for the production of (R)-1-phenylethane-1,2-diol increased from 17 for the wild-type enzyme to 91, as well as twofold-improved activity for the production of (R)-1-phenylethane-1,2-diol (1.96 ± 0.09 versus 1.04 ± 0.07 μmol/min/mg for wild-type EchA). Computer modeling indicated that this mutation significantly alters (R)-styrene oxide binding in the active site. Another three variants from EchA active-site engineering, F108L/C248I, I219L/C248I, and F108L/I219L/C248I, also exhibited improved enantioselectivity toward racemic styrene oxide in favor of production of the corresponding diol in the (R) configuration (twofold enhancement in their E values). Variant F108L/I219L/C248I also demonstrated 10-fold- and 2-fold-increased activity on 5 mM epoxypropane (24 ± 2 versus 2.4 ± 0.3 μmol/min/mg for the wild-type enzyme) and 5 mM 1,2-epoxyhexane (5.2 ± 0.5 versus 2.6 ± 0.0 μmol/min/mg for the wild-type enzyme). Both variants L190F (isolated from a DNA shuffling library) and L190Y (created from subsequent saturation mutagenesis) showed significantly enhanced activity for racemic styrene oxide hydrolysis, with 4.8-fold (8.6 ± 0.3 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) and 2.7-fold (4.8 ± 0.8 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) improvements, respectively. L190Y also hydrolyzed 1,2-epoxyhexane 2.5 times faster than the wild-type enzyme.